Immune System and T Cell Epitopes
Immune responses to biological therapeutic agents are wide ranging, and can be directed against agents that are both non-human and human in origin. These responses include those that elicit a weak clinical effect and those that limit efficacy which can occasionally result in morbidity or even mortality in patients. In particular, serious complications can arise with the production of neutralizing antibodies, especially when they target recombinant self proteins and therefore have the potential to cross react with the patient's own endogenous protein (Lim, 2005). Problems associated with immunogenicity to biologics (i.e., therapeutic medical products; such as, antibodies and recombinant proteins/polypeptides) have been reduced largely due to advances in molecular biology. There are, however, many recombinant protein biologics that are identical to endogenously expressed human sequences that still elicit potent neutralizing immune responses in patients (Hochuli, 1997; Schellekens et al, 1997; Namaka et al, 2006). The mechanism by which immunogenicity is triggered remains unclear although the tolerance to self proteins may be broken by a number of factors linked to both the product and the patient (reviewed in Chester et al, 2006; Baker and Jones, 2007). For the product, these include dose, frequency of administration, route, immunomodulatory capacity of the protein therapeutic, and the formulation (Jaber and Baker, 2007). For the patient, factors such as immune competence (i.e. whether the patient is receiving immunosuppressive treatment), patient's MHC haplotype and intrinsic tolerance to the protein therapeutic will influence immunogenicity. Regardless of how immunogenicity is triggered, one of the single most important factors in the development of an ensuing immune response is the presence of epitopes that are able to effectively stimulate a potent CD4+ T cell response (reviewed Baker and Jones, 2007).
T cells or T lymphocytes are a subset of white blood cells known as lymphocytes. (The abbreviation “T” in T cell is for “thymus” since this is the primary organ responsible for T cell maturation.) T cells play a central role in cell-mediated immunity. They can be distinguished from other types of lymphocytes (such as B cells and natural killer cells (NK cells)), by the presence of cell-surface proteins called T cell receptors (TCRs). Different types of T cells have also been identified; these can be distinguished based on the differing functions they serve (e.g., CD4+ T cells (a.k.a., TH or T helper cells), CD8+ cytotoxic T cells (CTLs), memory T cells, regulatory T cells (Treg cells), natural killer cells (NK cells), and gamma delta T cells (γδ T cells)).
T helper (TH) cells are so named because they aid other white blood cells in immunologic processes including, inter alia, assisting the maturation of B cells into plasma and B memory cells, and activation of cytotoxic T cells and macrophages. TH cells are also known as CD4+ T cells because they express CD4 protein on the cell-surface. CD4+ T cells are activated when peptide antigens are presented by MHC class II molecules expressed on the surface of Antigen Presenting Cells (APCs). Once activated, CD4+ T cells divide rapidly and secrete chemokines that further assist in activating or regulating immune responses.
T cell epitope analysis is becoming increasingly important particularly in the pre-clinical analysis of biologics and may, in time, become a requirement for regulatory approval for clinical trials. To this end, a pre-clinical ex vivo T cell assay (EPISCREEN™) has been used to provide an effective technology for predicting T cell immunogenicity by identifying linear T cell epitopes present in protein sequences. Synthetic overlapping peptides typically of about 15 amino acids in length are tested against a cohort of community blood donors carefully selected based on MHC class II haplotypes to provide a quantitative analysis of T cell epitopes present in protein sequences. This technology has been used successfully to compare protein variants for the potential to induce an immune response in vivo. By providing a high degree of sensitivity along with high reproducibility, the EPISCREEN™ assay allows an accurate pre-clinical assessment of the potential for immunogenicity of biologics. See, Baker & Carr, “Preclinical Considerations in the Assessment of Immunogenicity for Protein Therapeutics,” Current Drug Safety 5(4):1-6 (2010); Bryson et al., “Prediction of Immunogenicity of Therapeutic Proteins: Validity of Computational Tools,” Biodrugs 24(1)1-8 (2010); Holgate & Baker, “Circumventing Immunogenicity in the Development of Therapeutic Antibodies,” IDrugs 12(4):233-237 (2009); Perry et al., “New Approaches to Prediction of Immune Responses to Therapeutic Proteins during Preclinical Development,” Drugs R D 9(6):385-396 (2008); and, Baker & Jones, “Identification and removal of immunogenicity in therapeutic proteins,” Current Opinion in Drug Discovery & Development 10(2):219-227 (2007).
Pseudomonas Exotoxin A
Pseudomonas exotoxin A (PE-A) is a highly potent, 66 kD, cytotoxic protein secreted by the bacterium Pseudomonas aeruginosa. PE-A causes cell death by inhibiting protein synthesis in eukaryotic cells via inactivation of translation elongation factor 2 (EF-2), which is mediated by PE-A catalyzing ADP-ribosylation of EF-2 (i.e., transfer of an ADP ribosyl moiety onto EF-2). PE-A typically produces death by causing liver failure.
PE-A has at least three different structural domains responsible for various biological activities (FIG. 1). See e.g., Siegall et al., Biochemistry, vol. 30, pp. 7154-7159 (1991); Theuer et al., Jour. Biol. Chem., vol. 267, no. 24, pp. 16872-16877 (1992); and, U.S. Pat. No. 5,821,238. PE-A domain IA (amino acids 1-252 (see e.g., SEQ ID NO:133)) is responsible for cell binding. Domain II (amino acids 253-364 (see e.g., SEQ ID NO:133)) is responsible for translocation of PE-A into the cell cytosol. Domain III, the cytotoxic domain (amino acids 400-613 (see e.g., SEQ ID NO:133)), is responsible for ADP ribosylation of Elongation Factor 2 (EF2); which thereby inactivates EF2, subsequently causing cell death. Additionally, a function for domain IB (amino acids 365-399 (SEQ ID NO:139)) has not been established. Indeed, it has been reported that amino acids 365-380 (SEQ ID NO:138) within domain IB can be deleted without producing an identifiable a loss of function. See, Siegall et al., Biochemistry, vol. 30, pp. 7154-7159 (1991).
It has also been reported that PE-A may comprise any one of at least three different carboxy-terminal tails (FIG. 1); these appear to be essential for maintaining or recycling proteins into the endoplasmic reticulum. See, Theuer et al., J. Biol. Chem., vol. 267, no. 24, pp. 16872-16877 (1992); Chaudhary et al., Proc. Natl. Acad. Sci. USA, vol. 87, pp. 308-312 (1990); and, Seetharam et al., Jour. Biol. Chem., vol. 266, 17376-17381 (1991). In particular, in correspondence with the exemplary sequence shown in FIG. 1 (SEQ ID NO:133) these alternative carboxy-terminal tails comprise amino acid sequences:
609-REDLK-613 (SEQ ID NO:135);
609-REDL-612 (SEQ ID NO:136); and
609-KDEL-612 (SEQ ID NO:137).
Variants of PE-A, modified to lack the cell binding domain but coupled to heterologous cell-specific targeting molecules (e.g., antibodies), have been shown to have reduced levels of non-specific toxicity. See e.g., U.S. Pat. No. 4,892,827.
Various forms of PE-A (e.g., truncated/deletion forms with molecular weights of ˜37 kD, 38 kD, 40 kD, et cetera) have been combined with a number of growth factors, antibodies, and other proteins to generate cytotoxins which selectively target cells of a desired phenotype. See, for example:                Kreitman et al., “Recombinant immunotoxins and other therapies for relapsed/refractory hairy cell leukemia,” Leuk. Lymphoma, Suppl. 2:82-86 (June-2011);        Itoi et al., “Targeting of locus ceruleus noradrenergic neurons expressing human interleukin-2 receptor α-subunit in transgenic mice by a recombinant immunotoxin anti-Tac(Fv)-PE38,” J. Neurosci., 31(16):6132-6139 (April-2011);        Shapira et al., “An immunoconjugate of anti-CD24 and Pseudomonas exotoxin selectively kills human colorectal tumors in mice,” Gastroenterology, 140(3):935-946 (March-2011);        Kuan et al., “Affinity-matured anti-glycoprotein NMB recombinant immunotoxins targeting malignant gliomas and melanomas,” Int. J. Cancer, 129(1):111-21 (July-2011);        Hu et al., “Investigation of a plasmid containing a novel immunotoxin VEGF165-PE38 gene for antiangiogenic therapy in a malignant glioma model,” Int. J. Cancer, 127(9):2222-2229 (November-2010);        Mareeva et al., “A novel composite immunotoxin that suppresses rabies virus production by the infected cells,” J. Immunol. Methods, 353(1-2):78-86 (February-2010);        Zielinski et al., “Affitoxin—a novel recombinant, HER2-specific, anticancer agent for targeted therapy of HER2-positive tumors,” J. Immunother. 32(8):817-825 (October-2009);        Theuer et al., J. Biol. Chem., 267(24):16872-16877 (1992);        Pastan et al., “Recombinant toxins for cancer treatment,” Science, 254:1173-1177 (1991);        U.S. Pat. No. 5,821,238 (“Recombinant Pseudomonas Exotoxins with Increased Activity”); and        U.S. Pat. No. 4,892,827 (“Recombinant Pseudomonas Exotoxins: Construction of an Active Immunotoxin with Low Side Effects”).        
A significant disadvantage in using PE-A for treatment of disease, however, is that it is a foreign (non-self) protein being introduced into a heterologous host (e.g., a human). Introduction of non-self proteins into heterologous hosts commonly elicits host immune reactions, such as the generation of antibodies (“neutralizing antibodies”) or immune cell reactions (e.g., cytotoxic T cell responses) which are directed at eliminating the non-self protein (i.e., PE-A). Accordingly, it would be advantageous if elements of PE-A (PE-A epitopes) which are recognized and targeted as “non-self” could be removed prior to use of this molecule as a therapeutic agent.
Deimmunization of PE
Some investigators have previously attempted to identify and remove immunogenic determinants from PE-A (i.e., to “deimmunize” PE-A). See, for example:                Pastan et al., “Immunotoxins with decreased immunogenicity and improved activity,” Leukemia and Lymphoma, 52(S2):87-90 (June-2011);        Onda et al., “Recombinant immunotoxin against B-cell malignancies with no immunogenicity in mice by removal of B-cell epitopes,” Proc. Natl. Acad. Sci. USA, 108(14):5742-5747 (April-2011);        Hansen et al., “A recombinant immunotoxin targeting CD22 with low immunogenicity, low nonspecific toxicity, and high antitumor activity in mice,” J. Immunother. 33(3):297-304 (April-2011);        Stish et al., “Design and modification of EGF4KDEL 7Mut, a novel bispecific ligand-directed toxin, with decreased immunogenicity and potent anti-mesothelioma activity,” Br. J. Cancer, 101(7):1114-1123 (October-2009);        Nagata et al., “Removal of B cell epitopes as a practical approach for reducing the immunogenicity of foreign protein-based therapeutics,” Adv. Drug Deliv. Rev., 61(11):977-985 (September-2009);        Onda et al., “An immunotoxin with greatly reduced immunogenicity by identification and removal of B cell epitopes,” Proc. Natl. Acad. Sci. USA, 105(32):11311-11316 (August-2008); and        Pastan et al, “Mutated Pseudomonas Exotoxins with Reduced Antigenicity,” U.S. Patent Application No. 2009/0142341.        
Despite progress in the area of deimmunization of PE-A, there remains a need for the development of optimized, less immunogenic or non-immunogenic, biologically active forms of this useful cytotoxin. The invention described herein addresses this need.